The major aim of this project is to investigate mechanisms of cellular Ca regulation in liver. The focus of these experiments will be (a) to define the physiological range of mitochondrial Ca concentrations and the role of Ca2+ transport in mitochondria, specifically to study the hypothesis that it serves to regulate matrix free Ca2+ and (b) to identify the major intracellular source(s) of activator Ca2+, i.e., Ca stores mobilized upon stimulation with vasopressin or alpha adrenergic agonists, resulting in an increase in cytoplasmic Ca2+ and activation of key enzymes such as glycogen phosphorylase. The results of these studies will contribute to a general understanding of stimulus-secretion coupling in other epithelial tissues, where Ca2+ is thought to play an essential role, such as acinar cells of the exocrine pancreas, and in Beta-cell insulin secretin. Total Ca concentration in mitochondria, endoplasmic reticulum (ER), and nuclein will be measured by electron probe microanalysis (EPMA) in parenchymal cells of rat liver, rapidly frozen in situ to preserve cell morphology and ion distribution. Subcellular Ca distribution will be measured in cells from stimulated and unstimulated liver to determine the range of Ca concentrations in mitochondria and ER under physiological conditions. The combined techniques to be used in this study i.e., a new in situ rapid freezing technique, cryoultramicrotomy and EPMA, constitute a unique and powerful approach for directly measuring the intracellcular Ca distribution in situ.